八肋游仆虫中心蛋白的DNA结合性质与切割活性

DNA binding and cleavage studies of Euplotes octocarinatus centrin

为了探究中心蛋白在核苷酸切除修复识别中的可能作用,选择八肋游仆虫中心蛋白(EoCen)作为研究对象,采用荧光光谱,圆二色光谱,等温滴定量热(ITC)以及电泳等方法研究了EoCen与DNA之间的相互作用.结果表明,在室温下pH为7.4的10 mmol·L^(-1)N-2-羟乙基哌嗪-N′-乙磺酸(hepes)缓冲溶液中,EoCen与DNA的结合导致EoCen二级结构发生改变,α-helix含量减小,DNA双螺旋结构发生微扰.同时DNA的结合会抑制EoCen的聚集.荧光光谱与ITC分析发现,EoCen与DNA以化学计量比1∶1结合形成配合物,结合常数约为10^(4) L·mol^(-1).琼脂糖凝胶电泳分析表明EoCen具有切割活性,能够将超螺旋质粒DNA首先切成缺口型,最后变成线型.

Centrin is ubiquitous in all eukaryote species and plays important roles in various cellular processes.Recently research has reported that centrin is strongly associated with a recognition process in nucleotide excision repair.In order to explore the possible role of centrin in the process,the interaction of Euplotes octocarinatus centrin(EoCen)and DNA was characterized by using different methods including various spectroscopic techniques,isothermal titration calorimetry(ITC)and electrophoresis analyses.The results indicate that DNA can bind to EoCen with 1∶1 stoichiometry and the conditional binding constant is about 10^(4) L·mol^(-1)in 10 mmol·L^(-1)Hepes solution(pH=7.4)at room temperature as shown by ITC and fluoresence spectroscopy.The enthalpy change associated with DNA binding to EoCen is positive,indicative of an endothermic process.Circular dichroism(CD)and UV-Vis experiments reveal that the formation of EoCen-DNA complex results in a conformational change of protein and DNA.In more detail,the content ofα-helix of protein decreases and a certain perturbation takes place in the double helix structure of DNA.In addition,the binding to DNA is not in favour of the aggregation of proteins as demonstrated by native polyacrylamide gel electrophoresis(native-PAGE)assay.Subsequently,the DNA cleavage activity of EoCen is ascertained by gel electrophoresis assay.It is observed that DNA can be cleaved by EoCen after longer time incubation.DNA cleavage products do not appear in the presence of human serum albumin(HSA)under the similar conditions.It reveals that EoCen is a good DNA cleavage agent.DNA is converted from supercoiled form to nicked circular form and further conversion to linear form in the presence of EoCen.Cleavage efficiency of EoCen appears when the protein concentration is 0.85μmol·L^(-1).The increasingly stronger conversions of supercoiled form to nicked circular form and then linear form of DNA are observed with the increase of concentration of EoCen.Besides,the kinetic parameters in regard to the ability of EoCen to cleave DNA were calculated by following the time dependence of the reaction in 10 mmol·L^(-1)Hepes solution(pH=7.4)at room temperature.The loss of DNA of supercoiled form and increased levels of nicked circular form and linear form were quantified after gel electrophoresis,and then fitted with the aid of the single-exponential fitting procedures.The reaction profile of DNA cleavage for the loss of supercoiled form exhibits first-order kinetic behavior,with k=(0.0279±0.0020)min^(-1)of EoCen.The report provides valuable information for understanding the functional diversity of centrin.